Topoisomerase Inhibitors Modulate Gene Expression of B-Cell Translocation Gene 2 and Prostate Specific Antigen in Prostate Carcinoma Cells

Kun-Chun Chiang,Phei-Lang Chang,Ke-Hung Tsui,Li-Chuan Chung,Wen-Tsung Chen,Horng-Heng Juang,Chun-Nan Yeh,Natasha Kyprianou

doi:10.1371/journal.pone.0089117

Abstract

Camptothecin (CPT) and doxorubicin (DOX) have been demonstrated to have potent anti-tumor activity. The B-cell translocation gene 2 (BTG2) is involved in the regulation of cell cycle progression. We evaluated the molecular mechanisms of CPT and DOX on cell proliferation and the expressions of BTG2 and prostate specific antigen (PSA) in prostate carcinoma cells. Our results indicated that CPT or DOX treatments induced Go/G1 cell cycle arrest in LNCaP cells and apoptosis at higher dosage. Immunoblot and transient gene expression assay indicated that CPT or DOX treatments induced p53 and BTG2 gene expression, with the later effect dependent on the p53 response element within BTG2 promoter area since mutation of the p53 response element from GGGAAAGTCC to GGAGTCC or from GGCAGAGCCC to GGCACC by site-directed mutagenesis abolished the stimulation of CPT or DOX on the BTG2 promoter activity, which is also supported by our results that cotreatments of pifithrin-α, an inhibitor of p53 dependent transcriptional activation, blocked the induction of CPT or DOX on BTG2 gene expression. CPT or DOX also downregulated the protein expressions of androgen receptor (AR) and PSA. Transient gene expression assays suggested that CPT or DOX’s attenuation of PSA promoter activity is dependent on both the androgen and p53 response elements within of the PSA promoter. Our results indicated that CPT and DOX attenuate cell proliferation via upregulation of BTG2 gene expression through the p53-dependent pathway. The CPT and DOX block the PSA gene expression by upregulation of p53 activity and downregulation of androgen receptor activity.

Highlights

Prostate cancer, the second most common solid tumor for men in United States, has approximately 41,740 new cases diagnosed and 28,170 dying of this disease in 2012 [1]
Results from flow cytometric analysis of treated LNCaP cells revealed that low dose CPT (0– 1 mM) induced cell cycle arrest at G0/G1 dose-dependently. 1 mM CPT induced,20% increase in G0/G1 phase cell together with a decrease in S phase cells after 24 hours incubation in LNCaP cells (Figure 2B)
High dose of CPT (2 to 4 mM) induced cell apoptosis indicated by the 15–20% increase of sub-G1 fraction of cells (Figure 2B), which was supported by immunoblot assay result which revealed that treatment with 1–2 mM of CPT induced the expression of cleaved form of PARP in LNCaP cells (Figure 2C)

Summary

Introduction

The second most common solid tumor for men in United States, has approximately 41,740 new cases diagnosed and 28,170 dying of this disease in 2012 [1]. For the high-risk prostate cancer patients, accounting for 15% of all prostate cancer, 30–60% of them would develop biochemical recurrence (BCR), flare up of PSA, at around 10 years [2], followed by the clinical metastasis months to years later [3]. Even under the state-of-art management for high risk prostate cancer patients, which include multimodal approaches, 10–25% prostate cancer patients die of metastatic disease [3]. Topoisomerases, a family of highly conserved enzymes, play an important role during DNA topology and exist ubiquitously in all eukaryotic cells. Doxorubicin (DOX), the commonly used chemotherapeutic drug, exerts its antitumor effect through inhibiting TOP I and II to intervene in DNA uncoiling [5]

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Results

Conclusion